UBIQUITIN-CONJUGATING ENZYME 3 DELAYS HUMAN LENS EPITHELIAL CELLS IN METAPHASE

Authors: LIU, QING - TUFTS/HNRCA; Shang, Fu; WHITCOMB, ELIZABETH - TUFTS/HNRCA; GUO, WEIMIN - TUFTS/HNRCA; LI, WEI - TUFTS/HNRCA; Taylor, Allen

Submitted to: Investigative Ophthalmology and Visual Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/21/2005
Publication Date: 2/15/2006
Citation: Liu, Q., Shang, F., Whitcomb, E., Guo, W., Li, W., Taylor, A. 2006. Ubiquitin-conjugating enzyme 3 delays human lens epithelial cells in metaphase. Investigative Ophthalmology and Visual Science. 47(4):1302-9.

Interpretive Summary: The ubiquitin-conjugating enzyme (Ubc) Ubc3/Cdc4 has well established functions in the G1-to-S-phase transition, one of the phases in which new cells develop. The authors explored the roles for the ubiquitin-conjugating enzyme in regulating the human lens epithelial cell (HLEC) cycle and expect to find similar effects in human lens epithelial cells. Cell cycle progression was determined by the use of three types of ubiquitin-conjugating enzymes expressed in the HLECs by using an adenoviral vector, a DNA-containing virus used to transfer DNA into the cells. Expression of the ubiquitin-conjugating enzymes mt and wtUbc3 delayed the cell cycle in metaphase instead of the expected G1 phase. Expression of both Ubc3s also stabilized the M-phase regulators, cyclin A, cyclin B, and securin. The Ubc3 enzymes seemed to have played different roles from the functions that involve targeting G1/S regulators for degradation. Effects of inhibiting the proteasome on the cell cycle of HLECs were also directly investigated; when the proteasome inhibitor was added to S-phase cells, the M-phase regulators were stabilized and the cells were arrested in the G2/M phase of cell cycle proliferation, the growth of new cells. In contrast, if the proteasome inhibitor was added before the cells entered the S phase, the G1 kinase inhibitors p21WAF and p27KIP was observed to be stabilized and the cells were arrested in the G1 phase. In conclusion, although the ubiquitin-proteasome pathway is involved in regulating the transitions between all phases of the HLEC cycle, with the previously described roles that Ubc3 had in governing G1/S transitions, the expression of Ubc3 delays the HLEC cycle in the metaphase, or M-phase. As a result, the data suggests new and different roles for the Ubc3 enzymes that do not involve the transfer of ubiquitin in the M phase of the HLEC cell cycle.

Technical Abstract: Ubc3/Cdc34 is a ubiquitin-conjugating enzyme (Ubc) with well established functions in the G1-to-S-phase transition. Expecting to find similar effects in human lens epithelial cells (HLECs), the authors explored roles for this ubiquitin-conjugating enzyme in regulation of the HLEC cycle. Catalytically incompetent Ubc3 (C88S, L97S), wild-type (wt)Ubc3, and mutant (mt)Ubc2 (C93A) were expressed in HLECs, by using an adenoviral vector, and cell cycle progression was assessed. Expression of mt- and wtUbc3, but not empty virus or mtUbc2, delayed the cell cycle in metaphase, rather than the expected G1 phase. Expression of both Ubc3s also stabilized M-phase regulators, cyclin A, cyclin B, and securin. Thus, it appeared that the Ubc3 enzymes were playing roles different from canonical proteolytic functions in targeting G1/S regulators for degradation. We also directly investigated the effect of inhibiting the proteasome on the cell cycle of HLECs. When the proteasome inhibitor was added to S-phase cells, the M-phase regulators were stabilized, and the cells were arrested in the G2/M phase. In contrast, if the proteasome inhibitor was added before the cells entered the S phase, stabilization of the G1 kinase inhibitors p21WAF and p27KIP was observed and the cells were arrested in the G1 phase. The ubiquitin-proteasome pathway is involved in regulation of transitions between all phases of the HLEC cycle. However, in contrast with previously described roles for Ubc3 in governing G1/S transitions, expression of Ubc3 delays the HLEC cycle in metaphase. The data suggest novel roles for Ubc3 that do not involve the transfer of ubiquitin in the M phase in the HLEC cell cycle.